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Spectrophotometric determination of trace amounts of phosphate in water

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Article  in  Water Science & Technology · October 2012

DOI: 10.2166/wst.2012.468 · Source: PubMed

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 2653 © IWA Publishing 2012 Water Science & Technology | 66.12 | 2012

Spectrophotometric determination of trace amounts

of phosphate in water and soil
S. Ganesh, Fahmida Khan, M. K. Ahmed, P. Velavendan, N. K. Pandey
and U. Kamachi Mudali

ABSTRACT
S. Ganesh
A simple spectrophotometric method has been developed for the determination of phosphate
P. Velavendan
dissolved in soil and water. The method is based on the formation of phosphomolybdate with added N. K. Pandey
U. Kamachi Mudali
ammonium molybdate followed by reduction with hydrazine in acidic medium. Orthophosphate and Reprocessing Group,
Indira Gandhi centre for atomic research,
molybdate ions condense in acidic solution to give molybdophosphoric (phosphomolybdic) acid, Kalpakkam, 603102,
India
which upon selective reduction (perhaps with hydrazinium sulphate) produces a blue colour, due to
Fahmida Khan (corresponding author)
molybdenum blue of uncertain composition. The intensity of blue colour is proportional to the
Department of Chemistry,
amount of phosphate. If the acidity at the time of reduction is 0.5 M in sulphuric acid and National Institute of Technology,
GE Road, Raipur, 492010,
hydrazinium sulphate is the reductant, the resulting blue complex exhibits maximum absorption at India
E-mail: fkhan.chy@nitrr.ac.in
830 nm. The system obeys Lambert–Beer’s law at 830 nm in the concentration range of 0.5–5 μg/mL
M. K. Ahmed
of phosphate with a relative standard deviation (RSD) of 0.1% and correlation coefficient of 0.99. Nuclear Fuel Complex,
4 –1 –1
Molar absorptivity was determined to be 2.9 × 10 L mol cm at 830 nm. The method is also Hyderabad 500 062,
India
applicable for the determination of phosphate in nuclear reprocessing plants, medical science,
clinical science, agriculture, metallurgy and environmental science.
Key words | ion chromatography, molybdenum blue, phosphate, soil, spectrophotometry, water

INTRODUCTION

Phosphorus is the most abundant element on the surface
of the earth and is most commonly found as phosphate.
It plays an important role in biochemical processes and
is a key factor in the eutrophication of surface water
(McCarty et al. ). Plants and animals depend on phos-
phate to perform photosynthesis and to respire. Increased
phosphate concentrations are linked with increasing rates
of plant growth (Yaqoop et al. ). In soils, phosphate is
usually insoluble, due to it being bound to calcium, iron,
and aluminum, or incorporated into organic matter –
human and animal wastes or decaying matter. Usually
the concentration of phosphate in the water from low fer-
tilized soils or forest soils is only a few (μg/mL)
micrograms per mL (Kempers ). The analytical chem-
istry of phosphate is very important in many fields, for
example, medicine, clinical science, agriculture, metal-
lurgy and environmental science (Motomizu et al. ).
The determination of the phosphate in soil requires the
solubilization of phosphorus through the decomposition/
destruction of materials in soil containing mineral and organic
doi: 10.2166/wst.2012.468
phosphorus. The two most widely recognized procedures for
determination of phosphate in soil are the sodium carbonate
fusion method and the perchloric acid digestion method
(Mendham et al. ). In recent years large quantities of phos-
phate have been used in beverages, detergents, fertilizer and
also in sugar industries. Hence very sensitive analytical
methods are required for the determination of phosphate in
soil and water. Analytical methods have been proposed for
the determination of phosphate at higher concentrations
using titrimetry (Susic et al. ) and complexo-gravimetry
(Mamadal & Kundu ). At ppm levels of phosphate,
methods suggested include colorimetry (Krishnamurthy &
Suryanarayana ; Williams et al. ), atomic absorption
spectroscopy (Christian & Feldman ), flow injection analy-
sis (Motomizu & Mitsuko ), high-performance liquid
chromatography (HPLC) (Botker et al. ), ion chromato-
graphy (IC) (Ruiz-Calero & Galceran ; Nollet )
and spectrophotometry (Hayashi et al. ; Borissova &
Mitropolitska ; Motomizu et al. ; Gutschik ;
Smeller ; Mahadevaiah et al. ). Among such
 2654 S. Ganesh et al. | Spectrophotometric determination of phosphate in water and soil Water Science & Technology | 66.12 | 2012

methods, spectrophotometry involving molybdovanadate and Ion chromatographic conditions

ammonium molybdate is the most commonly used method. In
the spectrophotometric method using ammonium molybdate, Ion chromatograph: Metrohm modular Ion chromato-
different reductants such as tin (II) chloride (Yathirajam & graphy system
Dhamija ), ascorbic acid (Theodore ), hydrazine sul- Analytical column: Metrosep A Supp 5-250, 250 mm
phate (Mendham et al. ; Carter & Gregorich ), and L × 4.0 mm ID
1-amino-2-naphthol-4-sulfonic acid (Burton ) have been
Guard column: Metrosep A Supp 4/5
employed; some of these methods also involve complicated
Eluent concentration: 3.2 mM Na2CO3 þ 1.0 mM NaHCO3
and expensive equipment and need an extraction procedure
Eluent flow rate: 0.7 mL/min
(Botker et al. ) and such techniques are usually not avail-
Pressure: 13.5 MPa
able in common laboratories. Therefore, a standardized,
sensitive, simple spectrophotometric procedure to be used in Detector: Suppressed conductivity
routine process control analysis was required. Analytical mode: Isocratic
Injection loop: 20 μL
W
Temperature: Ambient (25 C)
Run time: 28 min
EXPERIMENTAL
Quantitation: Peak area
Reagents

Reagents used, namely potassium di-hydrogen phosphate,

ammonium molybdate and hydrazine sulphate, were of
analytical reagent grade obtained from M/s Sarabhai M
chemicals, Baroda, India. Water double distilled in glass
was used. To start the process 14.325 mg of (100 μg/mL)
potassium dihydrogen phosphate was weighed and trans-
ferred into a 100 mL standard measuring flask; it was
dissolved in water and then diluted to the mark with distilled
water. Then 1.7081 g of ammonium molybdate was dis-
solved in about 150 mL of warm water; a slightly milky
solution resulted which was cooled to room temperature.
It was then transferred into a 250 mL standard volumetric
flask and diluted to the mark with water. Then 0.125 g of
hydrazine sulphate was transferred into a clean 100 mL
beaker. It was dissolved in about 50 mL of water and then
the solution was transferred into a 100 mL standard measur-
ing flask. The beaker was washed three to four times with
water and washings were also transferred into the flask
and the solution was diluted to the mark with water.
Nitric acid (3.0 M) was prepared by suitable dilution of con-
centrated nitric acid (∼16 M) with double distilled water.
Instrumentation
A UV-VIS Spectrophotometer UV5704SS was used for
absorbance measurements. The wavelength range of this
spectrophotometer is 340–900 nm. All the absorbance
measurements were recorded by placing the sample tube
containing the solution.
PROCEDURE
To suitable aliquots of stock standard solution, 1 mL of
ammonium molybdate and 0.4 mL of hydrazine sulphate
were added and the solution was made up to 10 mL with
double distilled water in a standard measuring flask. The
standard measuring flasks were kept in a water bath for heat-
ing for 30 min. The temperature of the water bath was set to
W
60 C. While heating, a blue colour develops due to the for-
mation of ammonium phosphomolybdate complex. After
heating for 30 min the solution was cooled and its
absorbance was measured at wavelength 830 nm. An exper-
imental blank solution was used for carrying out correction
for the baseline.
Water and soil samples were collected from various
locations in Chhattisgarh State, India. A water sample was
filtered through Whatman filter paper and collected. Ali-
quots of the sample were used for its phosphate analysis.
Soil samples were digested, filtered and used for phosphate
analysis. In the perchloric digestion method, 2 g of accu-
rately weighed soil sample was taken in a 250 mL
Erlenmeyer flask and heated in a hot plate or by using an
aluminum block digestor with a 250 mL digestion tube,
then 20 mL of concentrated nitric acid was added into the
flask and mixed well. To achieve oxidation, the organic
W
matter in the sample was heated to approximately 130 C.
Organic matter oxidation was complete when the dark
colour due to organic matter in the sample disappeared.
The soil-HNO3 mixture was allowed to cool slightly. In
 2655 S. Ganesh et al. | Spectrophotometric determination of phosphate in water and soil
the HClO4 fume hood, 30 mL HClO4 was added to the
sample at boiling temperature ∼200 C for 20 min. During
W
that time dense white fumes appear and the insoluble solid
material is left in the bottom of the flask. When necessary,
a little extra HClO4 was used to wash down any black par-
ticles that had stuck to the sides of the flask. After
allowing the mixture to cool for another 10–15 min, the mix-
ture was transferred to a 250 mL volumetric flask and made
up to the mark with distilled water. Sediment was allowed to
settle before taking the aliquot for analysis.
From these solutions, a suitable aliquot was taken and
1 mL of ammonium molybdate and 0.4 mL of hydrazine
sulphate were added, it was made up to the mark with
water in a 10 mL flask and heated for 30 min in a water
W
bath maintained at 60 C. After blue colour developed,
the solution was cooled and its absorbance measured at
830 nm as mentioned above. All solutions, namely exper-
imental blanks, standards and unknown samples were
run in duplicate.
Ion chromatography
In order to compare the results obtained by spectrophoto-
metric analysis with those of the ion chromatographic
technique, experiments were conducted for the determi-
nation of phosphate present in environmental samples by
the IC technique using a conductivity detector. Various
phosphate standards of known concentrations (0.1–10 μg/
mL) were prepared by dissolving standard potassium di-
hydrogen phosphate (KH2PO4) in Millipore water. Twenty
microlitres of these solutions were injected directly into
the IC column connected to the conductivity detector. A
mixture of 3.2 mM sodium carbonate and 1.0 mM sodium
bicarbonate solution is used as a mobile phase with a flow
rate of 0.7 mL/min. A calibration graph was made for the
concentration range of inorganic phosphate from 0.1 to
10 μg/mL with an RSD of 2.239% and a correlation coeffi-
cient of 0.9994. Unknown samples were filtered and 20 μL
injected directly into the IC column for the determination
of phosphate concentration.
RESULTS AND DISCUSSION
The developed method is based on the formation of phos-
phomolybdate complex due to the reaction between
molybdate and phosphate, followed by its reduction with
hydrazine sulphate in aqueous acidic medium. Orthopho-
sphate and molybdate ions condense in acidic solution to
Water Science & Technology | 66.12 | 2012
give molybdophosphoric (phosphomolybdic) acid, which
upon selective reduction (perhaps with hydrazinium sul-
phate) produces a blue colour, due to molybdenum blue of
uncertain composition. The intensity of blue colour is pro-
portional to the amount of phosphate initially
incorporated in the heteropolyacid. If the acidity at the
time of reduction is 0.5 M in sulphuric acid and hydrazi-
nium sulphate is the reductant, the resulting blue complex
exhibits maximum absorption at 820–840 nm (Carter &
Gregorich ):
þ 72Hþ ! 7[PMo12 O40 ]
6 3
7PO3
4 þ 12[Mo7 O24 ] þ 36H2 O
3
2[PMo12 O40 ] ! P2 O5  24MoO3 þ 6NH3 ↑ þ3H2 O
Controlled heating in a water bath alone provided the
uniform colour development and consistent results. Under
optimized experimental conditions, with fixed concen-
tration of molybdate and reducing agent, the colour
intensity was found to be proportional to the amount of
phosphate present in disodium hydrogen phosphate. The
reaction conditions, as well as the various experimental par-
ameters affecting the formation and stability of the coloured
complex, were carefully investigated and optimized for
quantitative determination of phosphate in various samples.
The experimental variables such as concentration of
ammonium molybdate and concentration of reducing
agent, order of addition of the reagents and also colour stab-
ility of the complex were optimized for the effective
determination of phosphate (Ganesh et al. ). Figure 1
provides the data on the optimum quantity of 0.7%
Figure 1 | Effect of ammonium molybdate.
 2656 S. Ganesh et al. | Spectrophotometric determination of phosphate in water and soil Water Science & Technology | 66.12 | 2012

ammonium molybdate needed for effective determination of

phosphate. In this experiment, the different volumes of
ammonium molybdate were added to the solution contain-
ing a constant volume of 0.4 mL hydrazine sulphate and
1.3 ppm of phosphate. From the figure, it was obvious that
1 mL of 0.7% ammonium molybdate is enough to determine
the presence of phosphate. When higher volumes of
ammonium molybdate were added, there was no change
in absorbance. The maximum volume of hydrazine sulphate
required for the determination of phosphate is presented in
Figure 2. From this it was found that 0.4 mL of 0.12% hydra-
zine sulphate is sufficient for reduction and even if the
volume of hydrazine sulphate is increased beyond 0.4 mL
this has no effect on the phosphate estimation. The colour
is stable with no difference in absorbance being detectable
when phosphate standards were kept for up to 24 h. Figure 3
Figure 3 | Calibration graph for phosphate ion at 830 nm.
shows the calibration graph obtained at the wavelength of
maximum absorption at 830 nm for standard phosphate sol-
utions. At this wavelength the system obeys Lambert–Beer’s Table 1 | Typical results of phosphate in soil

law in the concentration range of 0.5–5.0 μg/mL of phos-

S. No Absorbance Conc. of phosphate (μg/mL)
phate. Molar absorptivity was determined to be 2.9 × 104
S-1 0.892 2.942
L mol–1 cm–1. Results obtained by using this methodology
and instrumentation for the determination of phosphate in S-2 0.928 3.061

soil/water samples are reported in Tables 1 and 2. The S-3 0.609 2.001
results obtained are reproducible with a standard deviation S-4 0.525 1.732
of 0.1% and correlation coefficient of 0.9995. The metal S-5 0.198 0.637
ions such as Mg2þ, Ca2þ, Mn2þ, Co2þ, Ni2þ, Zn2þ, Cd2þ, S-6 0.325 1.072
Pb2þ, Hg2þ, Fe3þ, Al3þ, Cr3þ and UO2þ 2 do not interfere S-7 0.389 1.283
with the determination of phosphate in concentrations up S-8 0.274 0.904
to 100 μg/mL. The presence of more than 500 molar S-9 0.468 1.544
amounts of As5þ can be tolerated by adding hydrazine S-10 0.555 1.831

Soil samples were collected from various sites located in Chhattisgarh state, India.

Table 2 | Typical results of phosphate in water

S. No Absorbance Conc. of phosphate (μg/mL)

W1 0.411 1.356
W2 0.292 0.963
W3 0.310 1.022
W4 0.642 2.118
W5 0.788 2.600
W6 0.175 0.577
W7 0.243 0.802
W8 0.364 1.201
W9 0.511 1.685
W10 0.812 2.678

Figure 2 | Effect of hydrazine sulphate. Water samples were collected from various sites located in Chhattisgarh state, India.
 2657 S. Ganesh et al. | Spectrophotometric determination of phosphate in water and soil Water Science & Technology | 66.12 | 2012

sulphate solution (Ohashi et al. ). The accuracy of the Table 3 | Comparison of two different analytical techniques

present method was checked by determining phosphate in

Concentration of phosphate (μg/mL)
various samples by both the present method and by an inde-
pendent technique, namely, IC. A typical chromatogram and Ion-chromatographic
S. No. Present method technique
a calibration curve are shown in Figures 4 and 5. The results
obtained for phosphate by the proposed method and by the 1 3.588 3.609
IC method agreed well within the limits of experimental 2 1.080 1.107
error and are reported in Table 3. 3 4.111 4.230
4 2.218 2.275
5 0.803 0.717
CONCLUSION 6 0.140 0.132

A modified spectrophotometric method for the determi-

nation of phosphate in soil is proposed. It is simple, fast of phosphate in sediments, soil, plants and ores as well as in
and accurate. On the basis of recent investigations, this biological materials such as urine and blood serum.
method shows potential for application to the determination

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